Gene Expression and Signal Transduction in Transformatio

转化中的基因表达和信号转导

• 批准号: 7337956
• 负责人: J F MUSHINSKI
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7337956
• 关键词: Gene; Expression; Signal; Transduction; Transformatio

The chief objective of our research is to understand the molecular and genetic mechanisms responsible for differentiation, cell growth, and neoplastic transformation. We study the oncogenes, tumor-suppressor genes and signal-transducing proteins in mouse and human experimental tumor systems, including BALB/c mouse plasmacytomas, B-cell lymphomas, and NIH 3T3 cells, among others. These are valuable experimental models, because they can be used to devise more specific therapy and preventive measures for human multiple myeloma, non-Hodgkin's lymphomas, and other human malignancies. BALB/c plasmacytomas, like human Burkitt lymphomas, are characterized by constitutive expression of the proto-oncogene, c-Myc. To determine which additional genetic alterations are required for complete transformation, we are using microarray hybridization studies of global gene expression to follow changes in gene expression during progression from pre-malignant to fully malignant plasma cell tumors. We are also using microarray hybridization studies to probe the molecular mechanisms at work in development of plasma cell tumors in mice and the mechanisms whereby certain transgenes and viral oncogenes accelerate this neoplastic process. Global gene expression studies are also underway to determine the physiological changes necessary for these tumors to adapt to growth in tissue culture. It is our hypothesis that such adaptive changes in gene expression that enable tumor cells to grow in the foreign environment of culture vessels might be analogous to those needed for human tumors to grow in alien environments following invasion or metastasis.In the study of signal transduction in differentiation and neoplastic transformation, we are investigating the isoform-specific features of the protein kinase C (PKC) family of serine/threonine kinases. We have been focusing on the delta and epsilon isoenzymes, which have opposing effects on cell proliferation. We have shown that most of the isoenzyme-specific determinants are located in the catalytic half (the carboxyl-terminal domain) of these PKCs by creating reciprocal chimeric cDNAs that encode molecules that are half PKC-delta and half PKC-epsilon. We are further dissecting the structure of the catalytic domain to determine which sub-domains determine PKC isoform- specific functions, focusing on the carboxy-terminal 50 amino acids, the "V5 domain." We are also studying the nature of PKC's involvement in apoptosis, in cytoskeleton-related changes in cell shape and motility, and in cooperation with the c-Myc proto-oncogene. We have shown that phorbol ester-activation of overexpressed PKC-delta disrupts the actin cytoskeleton in human and mouse lymphocytes, leading to the loss of membrane ruffling, a surface alteration needed for cell movement, and the loss of the typical elongated shape of these cells. We have demonstrated that this effect is due to PKC-mediated changes in phosphorylation of key tyrosine residues in the adaptor molecule, paxillin. Whereas the PKC-mediated effects on loss of tyrosine phosphorylation are indirect, we also have learned that PKC-delta can directly bind paxillin and phosphorylate a specific threonine, leading to homotypic aggregation.We have also shown that Myc and one of the PKC isoforms, PKC-gamma, can cooperate to transform NIH3T3 cells in vitro and in vivo, apparently not requiring intra-nuclear Myc. We are trying to understand the mechanism whereby this is accomplished.

我们研究的主要目标是了解负责分化、细胞生长和肿瘤转化的分子和遗传机制。我们研究小鼠和人类实验肿瘤系统中的癌基因、肿瘤抑制基因和信号转导蛋白，包括 BALB/c 小鼠浆细胞瘤、B 细胞淋巴瘤和 NIH 3T3 细胞等。这些都是有价值的实验模型，因为它们可用于为人类多发性骨髓瘤、非霍奇金淋巴瘤和其他人类恶性肿瘤设计更具体的治疗和预防措施。 BALB/c 浆细胞瘤与人伯基特淋巴瘤一样，其特征是原癌基因 c-Myc 的组成型表达。为了确定完全转化需要哪些额外的遗传改变，我们正在使用全局基因表达的微阵列杂交研究来跟踪从恶性前浆细胞肿瘤到完全恶性浆细胞肿瘤进展过程中基因表达的变化。我们还利用微阵列杂交研究来探讨小鼠浆细胞肿瘤发展的分子机制以及某些转基因和病毒癌基因加速这种肿瘤过程的机制。全球基因表达研究也在进行中，以确定这些肿瘤适应组织培养中生长所需的生理变化。我们的假设是，这种基因表达的适应性变化使肿瘤细胞能够在培养皿的外来环境中生长，这可能类似于人类肿瘤在侵袭或转移后在外来环境中生长所需的变化。分化和肿瘤转化，我们正在研究丝氨酸/苏氨酸激酶的蛋白激酶 C (PKC) 家族的亚型特异性特征。我们一直关注δ同工酶和ε同工酶，它们对细胞增殖具有相反的作用。我们通过创建编码一半 PKC-delta 和一半 PKC-epsilon 分子的相互嵌合 cDNA，证明大多数同工酶特异性决定簇位于这些 PKC 的催化半部分（羧基末端结构域）。我们正在进一步剖析催化结构域的结构，以确定哪些子结构域决定 PKC 同工型特异性功能，重点关注羧基末端 50 个氨基酸，即“V5 结构域”。我们还在研究 PKC 参与细胞凋亡、细胞骨架相关的细胞形状和运动变化以及与 c-Myc 原癌基因合作的性质。我们已经证明，佛波酯激活过表达的 PKC-δ 会破坏人和小鼠淋巴细胞中的肌动蛋白细胞骨架，导致膜皱褶消失、细胞运动所需的表面改变以及这些细胞典型的细长形状的丧失。我们已经证明，这种效应是由于 PKC 介导的接头分子桩蛋白中关键酪氨酸残基的磷酸化变化所致。虽然 PKC 介导的酪氨酸磷酸化损失的影响是间接的，但我们还了解到 PKC-delta 可以直接结合桩蛋白并磷酸化特定的苏氨酸，从而导致同型聚集。我们还表明 Myc 和 PKC 亚型之一， PKC-gamma，可以在体外和体内协同转化NIH3T3细胞，显然不需要核内Myc。我们正在尝试了解实现这一目标的机制。